Sensizitation of cancer cells to therapy using sina targeting genes from the 1p and 19q chromosomal regions

ABSTRACT

The invention relates to the identification of genes involved in resistance of cancer cells to therapy, to short nucleic acid molecules which inhibit the expression of these genes by RNA interference and to their use as adjuvant in cancer therapy, to sensitize cancer cells to conventional anticancer agents; the short nucleic acid molecules are double-stranded short interfering nucleic acid molecules including a sense and an antisense region, wherein the sense region includes a nucleotide sequence that is selected from the group consisting of: the sequences SEQ ID NO: 15, 11, 13, 14, 30, 31, 38, 46, 64 and 70 and the sequences having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with the sequences, and the antisense region includes a nucleotide sequence that is complementary to the sense region.

This application is a new divisional application of co-pendingapplication Ser. No. 12/375,960 filed on Nov. 16, 2009, which is a 371National stage application of PCT/IB07/03269 filed on Jul. 23, 2007,which claims priority to European Application No. 06291241.5 filed 5 onJul. 31, 2006. The entire contents of each of the above-identifiedapplications are hereby incorporated by reference.

The invention relates to the identification of genes involved inresistance of cancer cells to therapy, to short nucleic acid moleculeswhich inhibit the expression of these genes by RNA interference and totheir use as adjuvant in cancer therapy, to sensitize cancer cells toconventional anticancer agents.

Gliomas are the most prevalent primary brain tumours. Among them,astrocytomas are a notable source of preoccupation in oncology becausetheir incidence continually increases in industrialized countries(Ohgaki, H. and Kleihues, P., Acta Neuropathol., 2005, 109, 93-108) and,above all, their prognostic is pessimistic because they are refractoryto even the most aggressive therapy. For example, median survival timein a patient with glioblastoma multi-form (GBM), the worst grade ofastrocytoma, is between 6 to 15 months after diagnosis (Chinot, O. etMartin, P. M., Biologie des tumeurs cérébrales gliales, 1996,Montpellier, France). Standard treatment is surgery followed byradiotherapy and chemotherapy. Recently, a large phase III studydemonstrated that temozolomide (Temodal) therapy associated toradiotherapy provided a modest 3 months increase survival inglioblastoma (Stupp et al., N. Engl. J. Med., 2005, 352, 987-996).

Numerous works have addressed the chemoresistance as the principal causeof therapeutic fail (Harris, A. L., Int. J. Radiat. Biol. Relat. Stud.Phys. Chem. Med., 1985, 48, 675-680).

Contrasting with astrocytomas, oligodendrogliomas have a dramaticchemosensitivity (Perry et al., Arch. Neurol., 1999, 56, 434-436)resulting in a median survival of 10 years after medical management.Oligodendroglioma response to chemotherapy has been correlated with theloss of heterozygosity (LOH) on 1p and 19q chromosomal arms (Cairncrosset al., J. Natl. Cancer Inst., 1998, 90, 1473-1479) with a commonchromosomic area including 1p32-36 and 19q13.2-4 (Smith et al.,Oncogene, 1999, 18, 4144-4152).

The major part of studies addressing the chemoresistance of astrocytomasconcerned drug efflux mechanisms. However, the data are scattered andconflicting (Ashmore et al., Anticancer drugs, 1999, 10, 861-872).Expression of MDR-1 P-glycoprotein (P-gp), a protein associated withMDR, was not preferentially detected in resistant gliomas (Demeule etal., Int. J. Cancer, 2001, 93, 62-66). Moreover, MDR-type drugresistance in glioma cell lines results of long-term culturing, and invivo, only cerebral endothelium induces this mechanism (Bahr et al.,Brain Pathol., 2003, 13, 482-494). Finally, MDR-related genes were notlocated in the common 1p/19q deletion.

The correlation between 1p/19q LOH and oligodendrogliomaschemosensitivity strongly suggests that the reduction of globalexpression of some genes in the common region of deletion could beresponsible for the observed chemosensitivity.

An adjuvant treatment targeting these genes could increase thechemosensitivity of cancer cells.

The completion of human genome sequencing provides the opportunity toinvestigate potential candidates among the 1,700 genes located in the1p/19q LOH region.

Some studies have focused the search on genes with potential oncogenicproperties, which inactivation would lead to oligodendrogliomaoncogenesis but no convincing results were obtained to date.

Numerous genes located in the 1p/19q chromosomal regions (LOH regions)could have potential impact on drug resistance, including those involvedin drug efflux systems, metabolism, apoptosis, cell-cycle regulation andDNA-repair.

To date, the correlation between gene expression and drug resistance hasbeen demonstrated for two genes of the Nucleotide Excision Repair (NER)system, extensively studied in cancer cells: the ERCC1 and ERCC2 genes.Furthermore, a functional involvement of these genes in drug resistancehas been established for the ERCC1 gene only.

Chemotherapy drugs commonly employed, such as PCV regimen (procarbazine,lomustine or CCNU, and vincristine), cisplatin, fotemustine, ortemozolomide, are DNA-alkylating molecules. These drugs damage DNAleading cells to apoptosis. Cells can correct chemotherapy-inducedalterations thanks to various DNA-repair mechanisms (Li et al.,Anticancer Res., 2000, 20, 645-652; Wu et al., Clin. Cancer Res., 2003,9, 5874-5879) and thus overcome treatment. This phenomenom isresponsible for chemoresistance (Bosken et al., J. Natl. Cancer Inst.,2002, 94, 1091-1099).

A major role of ERCC1 in reparation of DNA alterations related toalkylating chemotherapy has been reported (Chaney S. G. & Sancar A., J.Natl. Cancer Inst., 1996, 88, 1346-1360; Li et al., Anticancer Res.,2000, 20, 645-652). Suppression of ERCC 1 expression in vitro byantisense or siRNA technology, leads to a decreased repair activity andan increased sensitivity of cultured cell lines to platinum-basedanticancer agents (Selvakumaran et al., Cancer Res., 2003, 63,1311-1316; Youn et al., Cancer Res., 2004, 64, 4849-4857; Chang et al.,Biochem. Biophys. Res. Commun., 2005, 327, 225-233).

ERCC2 (XPD) expression has been correlated with resistance to alkylatingcompounds in numerous cell lines (Chen et al., Ai Zheng, 2002, 21,233-239; Xu et al., Anticancer drugs, 2002, 13, 511-519), comprisingglioma cell lines (Chen et al., Neurosurgery, 1998, 42, 1112-1119) andit has been reported that its overexpression increased DNA repair inglioma cell lines (Chen et al., Chin. Med. J., 2003, 116, 1171-1174).However, a functional involvement of ERCC2 in drug resistance has notbeen established.

RNAi interference is the process where the introduction ofdouble-stranded RNA into a cell inhibits gene expression in a sequencedependent fashion (reviewed in Shuey et al., Drug Discovery Today, 2002,7, 1040-1046). RNAi has been observed in a number of organisms such asmammalian, Drosophila, nematodes, fungi and plants and is believed to beinvolved in anti-viral defense, modulation of transposon-activity andregulation of gene expression. RNAi is usually described as apost-transcriptional gene-silencing mechanism in which dsRNA triggersdegradation of homologous messenger RNA in the cytoplasm. Targetrecognition is highly sequence specific since one or two base pairmismatches between the siRNA and the target gene will greatly reducesilencing effect. The mediators of RNA interference are 21- and23-nucleotide small interfering RNAs (siRNA). In a second step, siRNAsbind to a ribonuclease complex called RNA-induced silencing complex(RISC) that guides the small siRNA to its homologous mRNA target.Consequently, RISC cuts the mRNA approximately in the middle of theregion paired with the antisens siRNA, after which the mRNA is furtherdegraded. Therefore, the use of exogenous siRNA holds great promise as anew tool for mammalian functional genomics and may also have futureapplications as gene-specific therapeutics.

Using siRNA technology, the inventors have established that four othergenes involved in DNA repair (MUTYH, PNKP, POLD1, and RUVBL2) and twogenes encoding P450 cytochrom isoforms (CYP2A6 and CYP4B1) are alsoinvolved in astrocytomas chemoresistance. In addition, the inventorshave established, for the first time that ERCC2 is functionally involvedin drug resistance.

MUTYH which belongs to the Base Excision Repair (BER) system is known torepair 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxodG) caused by oxidation.MUTYH mutations and variants were associated with development ofmultiple colorectal adenomas and cancers (Chow et al., Lancet Oncol.,2004, 5, 600-606). PNKP, also belonging to the BER system, was shown tobe involved in repairing DNA strand breaks caused by reactive oxygenspecies, ionizing radiations or alkylating agents (Whitehouse et al.,Cell, 2001, 104, 107-117; Chappell et al., EMBO J., 2002, 21,2827-2832). It has been related to susceptibility to genotoxic agentsbut not to chemoresistance (Rasouli-Nia et al., P.N.A.S., 2004, 101,6905-6910). POLD1 is known to be involved in NER and NMR (NucleotideMismatch Repair) systems, while RUVBL2 is known to be involved inhomologous recombination; these proteins have neither been related tochemosensitivity.

The cytochrome P450 isoforms CYP2A6 and CYP4B1 are known as activatorsof carcinogenic aromatic amines, and represent possible risk factors fortobacco-related and bladder cancers in human (Kamataki et al., Biochem.Res. Comm., 2005, Sep. 19; Imaoka et al., Biochem. Res. Comm., 2000,277, 776-780): they have neither been related to chemosensitivity.

The inventors have engineered siRNA which efficiently inhibit thetargeted genes expression and significantly sensitize astrocytoma cellsto chemotherapy.

These siRNAs are useful as adjuvant in cancer therapy, to sensitizecancer cells to chemotherapy and radiotherapy.

Therefore, the invention relates to a double-stranded short interferingnucleic acid (siNA) molecule comprising a sense and an antisense region,wherein the sense region comprises a nucleotide sequence that isselected from the group consisting of: the sequences SEQ ID NO: 11, 13,14, 15, 30, 31, 38, 46, 64 and 70 and the sequences having at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity with said sequences, and the antisense region comprises anucleotide sequence that is complementary to the sense region.

The siNA molecules according to the invention target seven genes (ERCC2,MUTYH, PNKP, POLD1, RUVBL2, CYP2A6 and CYP4B1) of the chromosomicregions 1p32-36 and 19q13.2-4 (loss of heterozygosity regions or LOHregion) which are all involved in resistance of cancer cells tochemotherapy and/or radiotherapy. The siNA molecules according to theinvention are able to down regulate the expression of the ERCC2, MUTYH,PNKP, POLD1, RUVBL2, CYP2A6 or CYP4B1 genes (target genes) by RNAinterference and thereby increase the sensitivity of cancer cells toconventional anticancer agents. Thus the siNA molecules according to thepresent invention potentiate the cytotoxic effect ofchemotherapy/radiotherapy on cancer cells.

The resistance of cancer cells to an anticancer agent may be evaluatedby a resistance index (RI) corresponding to the proportion of a cellpopulation that survived to treatment with said anticancer agent. It iscalculated as follows: cell number with anticancer agent treatment/cellnumber in control condition.

The sensibilization effect mediated by the siNA according to the presentinvention, may be evaluated by the siNA-induced drug sensibilizationindex (DS) which corresponds to the cell population (%) that survived toa simple treatment with an anticancer agent but died in response to thesame treatment with an siNA transfection. It is calculated as follows:(RI control siNA−RItarget siNA)/RIcontrol siNA×100; the target siNA isdirected to the resistance gene and the control siNA is directed to agene which is not involved in resistance to anticancer therapy.

Both indexes may be determined by any assay that measures cellviability, which is well-known in the art, such as for example a MTTassay.

Confirmation that the sensitization effect is mediated by inhibition ofthe target gene expression may be assayed by any RNA or protein analysistechnique, which is well-known in the art (Northern-blot, Western-blot,quantitative RT-PCR).

The siNA molecules according to the invention are defined by referenceto the human ERCC2, MUTYH, PNKP, POLD1, RUVBL2, CYP2A6 or CYP4B1 genesequences (Table I); the target sequence corresponds to the portion ofthe mRNA which is complementary to the antisense region of the siNAmolecule.

TABLE I Genes targeted with the SiNA GenBank Target accession genenumber Target sequence positions ERCC2 NM_000400 SEQ ID NO: 11 415-433SEQ ID NO: 13 1278-1296 SEQ ID NO: 14 1719-1737 SEO ID NO: 15 1978-1996MUTYH NM_012222 SEQ ID NO: 30 1475-1493 PNKP NM_007254 SEQ ID NO: 31379-397 POLD1 NM_002691 SEQ I NO: 38 1231-1249 RUVBL2 NM_006666 SEQ IDNO: 46 335-353 CYP2A6 NM_000762 SEQ ID NO: 64 591-609 CYP4B1 NM_000779SEQ ID NO: 70 645-663

The invention encompasses the synthetic, semi-synthetic or recombinantsiNA which inhibit the expression of a target gene from any organism.Given the positions of the targets in the human mRNAs, one skilled inthe art will easily find the corresponding positions in the homologoussequences of other organisms (eukaryotes, for example mammals) which areaccessible in the databases such as the NCBI database(http://www.ncbi.nlm.nih.gov/). Such homologous sequences can beidentified as is known in the art, for example using sequence alignment.In addition, the siNA molecule of the invention may inhibit theexpression of target gene variants, for example polymorphic variantsresulting from haplotype polymorphism.

siNA molecules can be designed to target such homologous sequences, forexample using perfectly complementary sequences or by incorporatingnon-canonical base pairs, for example mismatches and/or wobble basepairs, including flipped mismatches, single hydrogen bond mismatches,trans-type mismatches, triple base interactions and quadruple baseinteractions, that can provide additional target sequences. For example,the siNA molecule can be designed to target a sequence that is unique toa specific target gene mRNA sequence (a single allele or singlenucleotide polymorphism (SNP)) due to the high degree of specificitythat the siNA molecule requires, to mediate RNA activity. Alternatively,when mismatches are identified, non-canonical base-pairs (for examplemismatches and/or wobble bases) can be used to generate siNA moleculesthat target more than one sequence. In a non-limiting example,non-canonical base-pairs such as uu and cc base pairs are used togenerate siNA molecules that are capable of targeting homologous targetgene sequences. In this approach, a single siNA can be used to inhibitexpression of more than one gene instead of using more than one siNAmolecule to target the different genes.

DEFINITIONS

“short nucleic acid molecule” refers to a nucleic acid molecule no morethan 100 nucleotides in length, preferably no more than 80 nucleotidesin length, and most preferably, no more than 50 nucleotides in length.

“interfering nucleic acid molecule” refers to a nucleic acid moleculecapable of mediating RNA interference.“RNA interference” (RNAi) refers to the process of sequence specificpost-transcriptional gene silencing, induced by introduction of duplexesof synthetic short nucleic acid molecule in cells, for example duplexesof synthetic 21-nucleotide RNAs, as first described by Elbashir et al.,Nature 2001, 411, 494- and in the International PCT Application WO01/75164.“nucleotide” refers to standard ribonucleotides and deoxyribonucleotidesincluding natural bases (adenine, cytosine, guanine, thymine or uracil)and modified nucleotides that are modified at the sugar, phosphate,and/or base moiety.“Identity” refers to sequence identity between two nucleic acidmolecules. Identity can be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base, then themolecules are identical at that position. A degree of similarity oridentity between nucleic acid or amino acid sequences is a function ofthe number of identical or matching nucleotides at positions shared bythe nucleic acid sequences. Various alignment algorithms and/or programsmay be used to calculate the identity between two sequences, includingFASTA, or BLAST which are available as a part of the GCG sequenceanalysis package (University of Wisconsin, Madison, Wis.), and can beused with, e.g., default settings.“homologous” refers to a nucleic acid molecule having enough identity toanother one to lead to RNAi activity, more particularly having at least70% identity, preferably 80% identity and more preferably 90%.“complementary” refers to the ability of a nucleic acid to form hydrogenbond(s) by either traditional Watson-Crick base-pairing or othernon-traditional type base-pairing. In reference to the nucleic acidmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well-known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol., 1987, LII, pp 123-133, Frier et al., P.N.A.S., 1986,83, 9373-9377; Turner at al., J. Am. Chem. Soc., 1987, 109, 3783-3785).A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base-pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides, in thefirst oligonucleotide being base-paired to a second nucleic acidsequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90% and100% complementarity, respectively). “Perfectly complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence.“target gene” refers to a gene whose expression is to be down-regulated,e.g. ERCC2, MUTYH, PNKP, POLD1, RUVBL2, CYP2A6 or CYP4B1 gene.“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked.“anticancer agent”, “anticancer therapy” refers to both chemotherapyusing cytotoxic agents and radiotherapy.

In one embodiment, the invention features an siNA molecule wherein eachstrand comprises or consists of 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, andeach strand comprises at least 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)nucleotides that are complementary to the nucleotides of the otherstrand. For example, the siNA molecule of the invention comprises orconsists of a 19 to 21-nucleotide duplex (19 to 21 base pairs).

In another embodiment, the invention features an siNA molecule whereinthe sense region comprises or consists of a nucleotide sequence havingany of SEQ ID NO: 11, 13, 14, 15, 30, 31, 38, 46, 64 and 70 and theantisense region comprises or consists of a nucleotide sequence havingany of SEQ ID NO: 93 to 102, respectively. These siNA target the humangenes (Table II).

TABLE II siNA targeting the human genes   siNA sense strandsiNA antisense strand Target identification Identification  gene numbernumber sequence ERCC2 SEQ ID NO: 11 SEQ ID NO: 935′-gcauuucccaucgacgucc-3′ SEQ ID NO: 13 SEQ ID NO: 945′-ucaaagggcucgaugauga-3′ SEQ ID NO: 14 SEQ ID NO: 955′-auaaagagcagcuuguucc-3′ SEQ ID NO: 15 SEQ ID NO: 965′-aucgaagguaagaaaguca-3′ MUTYH SEQ ID NO: 30 SEQ ID NO: 975′-auauacuugauaugucagc-3′ PNKP SEQ ID NO: 31 SEQ ID NO: 985′-auugaccaaauacagugug-3′ POLD1 SEQ I NO: 38 SEQ ID NO: 995′-uggauguuguaaccgguga-3′ RUVBL2 SEQ ID NO: 46 SEQ ID NO: 1005′-ucaucuccagggagaagau-3′ CYP2A6 SEQ ID NO: 64 SEQ ID NO: 1015′-ugacaggaacucuuugucc-3′ CYP4B1 SEQ ID NO: 70 SEQ ID NO: 1025′-aucgcugacugcaagguag-3′

In another embodiment of the invention, the siNA molecule comprisesoverhanging nucleotide(s) at one or both end(s), preferably, 1 to about3 (e.g. about 1, 2, or 3) overhanging nucleotides. The overhangingnucleotides which are advantageously at the 3′ end of each strand, arepreferably 2′-deoxynucleotide(s), preferably 2′deoxypyrimidine(s), suchas a 2′-deoxythymidine(s). For example, the siNA molecule of theinvention is a 19 to 21-nucleotide duplex, with 19 to 21 base pairs with3′-terminal tt overhang(s).

In another embodiment of the invention, the siNA molecule comprisesblunt end(s), where both ends are blunt, or alternatively, where one ofthe ends is blunt.

In another embodiment of the invention, the siNA molecule is assembledfrom two separate oligonucleotide fragments or strands, wherein onefragment (sense strand) comprises the sense region and the secondfragment (antisense strand) comprises the antisense region of the siNAmolecule.

In another embodiment, the invention features an siNA molecule whereinthe sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. A nucleotidelinker can be a linker of at least 2 nucleotides in length, for exampleabout 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Examples of suchsiNA molecules include small hairpin nucleic acid (shNA) molecules.

A non-nucleotide linker comprises abasic nucleotides, aptamers,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds.

In another embodiment of the invention, the siNA molecule comprisesmismatches, bulges, loops or wobble base pairs to modulate the activityof the siNA molecule to mediate RNA interference.

In another embodiment of the invention, the siNA molecule comprises orconsists of ribonucleotide(s) (2′-OH nucleotides).

In addition, the siNA molecule may include one or more modificationswhich increase resistance to nuclease degradation in vivo and/or improvecellular uptake. The siNA may include nucleotides which are modified atthe sugar, phosphate, and/or base moiety, and/or modifications of the 5′or 3′ end(s), or the internucleotidic linkage.

In another embodiment of the invention, the siNA molecule comprises oneor more modified pyrimidine and/or purine nucleotides, preferably oneach strand of the double-stranded siNA. More preferably, said modifiednucleotides are selected from the group consisting of:2′-O-methylnucleotides, 2′-O-methoxyethylnucleotides, deoxynucleotides,such as 2′-deoxynucleotides and 2′-deoxy-2′-fluoronucleotides, universalbase nucleotides, acyclic nucleotides and 5-C-methyl nucleotides. AnsiNA molecule of the invention can generally comprise about 5% to about100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%modified nucleotides). The actual percentage of modified nucleotidespresent in a given siNA molecule will depend on the total number ofnucleotides present in the siNA molecule. The percent modification canbe based upon the total number of nucleotides present in the sensestrand, antisense strand or both the sense and the antisense strands.

In another embodiment, the invention features an siNA molecule whereinthe strand comprising the sense region (sense strand) includes aterminal cap moiety at the 5′-end, the 3′-end, or both the 5′ and 3′ends of the strand, preferably a deoxy abasic moiety or glyceryl moiety.

In another embodiment, the invention features an siNA molecule whereinthe strand comprising said antisense region (antisense strand) includesa phosphate group at the 5′-end.

In another embodiment of the invention, the siNA molecule comprises atleast one modified internucleotidic linkage, such as a phosphorothioatelinkage.

The siNA molecules according to the invention may be produced bychemical synthesis by using well-known oligonucleotides synthesismethods which make use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end and phosphoramidites, atthe 3′ end. The nucleic acid molecules of the present invention can bemodified to enhance stability by modification with nuclease resistantgroups, for example 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, TIBS, 1992, 17, 34 and Usman etal., Nucleic Acids Symp. Ser., 1994, 31, 163). Examples of such modifiedoligonucleotides include with no limitation: 2′ F-CTP, 2′ F-UTP, 2′NH2-CTP, 2′ NH2-UTP, 2′ N3-CTP, 2-thio CTP, 2-thio UTP, 4-thio UTP,5-iodo CTP, 5-iodo UTP, 5-bromo UTP, 2-chloro ATP, adenosine5′-(1-thiotriphosphate), cytidine 5′-(1-thiotriphosphate),guanosine-5′-(1-thiotriphosphate), uridine-5′-(1-thiotriphosphate),pseudo-UTP, 5-(3-aminoallyl)-UTP and 5-(3-aminoallyl)-dUTP. siNAcontructs can be purified by gel electrophoresis using general methodsor can be purified by high pressure liquid chromatography (HPLC) andre-suspended in water.

The chemically-synthesized siNA molecule according to the invention maybe assembled from two distinct oligonucleotides which are synthesizedseparately. Alternatively, both strands of the siNA molecule may besynthesized in tandem using a cleavable linker, for example asuccinyl-based linker.

Alternatively, the siNA molecules of the invention may be expressed (invitro or in vivo) from transcription units inserted into DNA or RNAvectors known to those skilled in the art and commercially available.

The invention relates also to a transcription unit comprising: atranscription initiation region, a transcription termination region, anda nucleic acid sequence encoding at least one siNA molecule according tothe present invention, wherein said nucleic acid sequence is operablylinked to said initiation region in a manner that allows expressionand/or delivery of the siNA molecule.

The nucleic acid sequence may encode one or both strands of the siNAmolecule, or a single self-complementary strand that self-hybridizesinto an siNA duplex.

The transcription initiation region may be from a promoter for aeukaryotic RNA polymerase I, II or III (pol I, II or III). Transcriptsfrom pol II or pol II promoters are expressed at high levels in allcells. Alternatively, prokaryotic RNA polymerase promoters may be used,providing that prokaryotic RNA polymerase enzyme is expressed in theappropriate cells. Transcription units derived from genes encoding U6small nuclear transfer RNA and adenovirus VA RNA are useful ingenerating high concentrations of desired siNA in cells.

The invention concerns also an expression vector comprising a nucleicacid encoding at least one siNA molecule of the instant invention. Theexpression vector may encode one or both strands of the siNA molecule,or a single self-complementary strand that self-hybridizes into an siNAduplex. The nucleic acid encoding the siNA molecule of the instantinvention is preferably inserted in a transcription unit as definedabove.

Large numbers of DNA or RNA vectors suitable for siNA moleculeexpression are known to those of skill in the art and commerciallyavailable. The recombinant vectors can be DNA plasmids or viral vectors.SiNA expressing viral vectors can be constructed based on, but notlimited to, adeno-associated virus, retrovirus, adenovirus oralphavirus. The recombinant vectors capable of expressing the siNAmolecules can be delivered in vivo, and persist in target cells.Alternatively, viral vectors can be used to provide transient expressionof siNA molecules.

The invention concerns also eukaryotic or prokaryotic cells which aremodified by a vector as defined above.

The invention concerns also a pharmaceutical composition comprising atleast an siNA molecule or an expression vector, as defined above, in anacceptable carrier, such as stabilizer, buffer and the like.

A pharmaceutical composition or formulation refers to a form suitablefor administration, e.g., systemic or local administration, into a cellor subject, including for example a human. Suitable forms, in part,depend upon the use or the route of entry, for example oral, inhalation,or by injection. These compositions or formulations are preparedaccording to any method known in the art for the manufacture ofpharmaceutical compositions.

In one embodiment, the invention features a composition wherein the siNAmolecule or vector is associated to a compound that allows the deliveryof the siNA/vector into cancer cells. The compound may be a membranepeptide, transporter, lipid, hydrophobic moiety, cationic polymer, PEI.Examples of membrane peptides include those able to cross theblood-brain barrier, such as with no limitation the Pep:Trans< >(http://www.syntem.com/english/techpeptrans.html). Preferably, the siNAand the compound are formulated in microspheres, nanoparticules orliposomes. Furthermore, the siNA molecule or vector may be associatedwith a compound that allows a specific targeting of the tumor, such as aligand of a cell-surface antigen or receptor, for example a peptide oran antibody specific for said antigen/receptor (e.g., PS100, PDGFR,erb-B2).

In another embodiment, the invention features a composition comprising acombination of at least two different siNA molecules.

In another embodiment, the invention features a composition wherein thesiNA molecule or vector is associated with at least one anticancer drug.

The invention also concerns an siNA molecule or a vector as definedabove, as a medicament.

The invention concerns also the use of an siNA molecule or a vector asdefined above, for the manufacture of a medicament for treating cancer.

The cancer may be of any type. Preferably, the cancer is a solid tumor,for example brain tumors such as astrocytomas, glioblastomas,oligodendrogliomas or mixed tumors.

In one embodiment of said use, the siNA molecule or vector is associatedwith an anticancer drug.

The invention concerns also a product containing at least one siNAmolecule or vector as defined above, and an anticancer drug, as acombined preparation for simultaneous, separate or sequential use inanticancer therapy.

The anticancer drugs which are used in combination with the siNAmolecule or the vector according to the invention are those commonlyused in chemotherapy, and include cytotoxic agents, such as alkylatingagents and antimetabolites.

Preferred anticancer drugs are alkylating agents, such as: cisplatin(cis-diaminedichloroplatinum, CDDP or DDP), temozolomide, fotemustine,procarbazine, lomustine and vincristine.

In addition, the siNA molecule according to the invention may be used incombination with other conventional anticancer therapies includingradiotherapy, immunotherapy and surgery.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence or treat (alleviate a symptom to some extent,preferably all the symptoms) of a disease or state. The pharmaceuticallyeffective dose of the siNA depends upon the type of cancer, thecomposition used, the route of administration, the type of mammal beingtreated, the physical characteristics of the specific mammal underconsideration, concurrent medication, and other factors, that thoseskilled in the medical arts will recognize. Generally, an amount between0.1 mg/kg and 100 mg/kg body weight/day of active ingredients isadministered.

The siNA of the invention may be administered by a single or multipleroute(s) chosen from: local (intratumoral, for example intracerebral(intrathecal, intraventricular)), parenteral (percutaneous,subcutaneous, intravenous, intramuscular, intraperitoneal), oral,sub-lingual, or inhalation.

When the siNA molecule or vector is used in combination withchemotherapy or radiotherapy, it is preferably administered immediatelyprior to the anticancer agent or several hours (2 to 48 hours) before.

In addition to the preceding features, the invention further comprisesother features which will emerge from the description which follows,which refers to examples illustrating the siNA molecules and their usesaccording to the invention, as well as to the appended drawings inwhich:

FIG. 1 illustrates the screening of DNA-repair genes in human glioma(GHD cells). Cells were seeded in 96-well plates, transfected with siRNAafter 24 h and treated with Cisplatin (CDDP) after 48 h. 96 hours posttreatment, alive cell number was evaluated with MTT. 1: ERCC2, 2:RAD54L, 3: LIG1, 4: MUTYH 5: PNKP, 6: POLD1, 7: REV7(MAD2L2), 8:MGC13170, 9: RUVBL2, G: GFP. The proportion of a cell population thatsurvives to chemotherapy is evaluated by the chemoresistance index (CI):cell number (OD) with chemotherapy/cell number (OD) in controlcondition. CI for each siRNA was compared with control CI (siRNA GFP).Data represented the mean of 3 independent experiments. CI is inferiorto CIGFP (0.5 or 50%) when siRNA transfection improves chemosensitivityand superior to CIGFP (grey) when it increases chemoresistance. Whitesquare corresponds to siRNA with low toxicity (<35% cell death withoutcisplatin) and with chemosensitization properties.

FIG. 2 illustrates drug sensitization by siRNAs targeting four DNArepair genes: ERCC1, ERCC2, MUTYH, and PNKP. SiRNAs were transfected in6 different astrocytoma derived cell lines treated with CDDP ortemozolomide (TMZ). DS corresponds to chemosensitivity induced by siRNA.siRNA targeting ERCC1 was included for comparison. Data represented themean of 3 independent experiments. Only significant results werereported (*p<0.05; **p<0.01).

FIG. 3 illustrates the effect of siRNAs targeting two cytochrome P450isoforms (CYP2A6 and CYP4B1) on the chemosensitization of astrocytomacells. SiRNAs were transfected in U373 cell line treated with CDDP. Thechemoresistance indice (CI) for each siRNA was compared with control CI(siRNA GFP). siRNA targeting ERCC1 was included for comparison. Datarepresented the mean of 3 independent experiments.

EXAMPLE 1 siRNA Directed to 1p/19q DNA Repair Genes are Able toSensitize Cancer Cells to Chemotherapy 1) Material and Methods a) CellCulture

U87, U373, U138, CCF and LN229 cells deriving from human astrocytomawere purchased from American Type Culture Collection (ATCC). GHD cellline derived from a human astrocytoma biopsy, was checked byfluorescence in situ hybridization, chromosome 7 polysomy, chromosome 10monosomy and immuno-histo-chemistry. Cells were maintained in DMEM(CAMBREX BIOSCIENCES), 10% FCS (v/v; ABCYS), and incubated in ahumidified atmosphere with 5% CO2, at 37 deg. C.

b) Inhibition of Gene Expression with siRNA

Three to five siRNA pairs were designed for each candidate gene andprepared into duplex form (EUROGENTEC). siRNAs targeting the GreenFluorescent Protein (GFP) and the ERCC 1 protein were used as controlsiRNAs. 24 hours after cell seeding (96 well plates), siRNA (150 nM)were transfected with Oligofectamine< > (Invitrogen), according to themanufacturer's instructions. Each condition (siRNA) was tested in 3independent experiments, each time in hexaplicate.

c) Cell Survival and Chemoresistance

Cells were seeded in 96 well plates, transfected with siRNA after 24 hand treated with cisplatine (CDDP, MERCK, 5 M final concentration) ortemozolomide (TMZ, SCHERING-PLOUGH, 1 M final concentration) after 48 h.Cell survival was determined 96 hours post-treatment, by measuringmitochondrial succinate dehydrogenase activity, with3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,SIGMA) added to a final concentration of 0.5 mg/ml into culture medium.Culture medium was discarded after 4 h of incubation with MTT andformazan crystals were dissolved in DMSO/ethanol (50/50, v/v). Opticaldensity was read at 540 nm. Chemoresistance was related to an index (CI)corresponding to the proportion of a cell population that survived tochemotherapy. It was calculated as follows: cell number (OD) withchemotherapy/cell number (OD) in control condition. The benefit of siRNAtransfection was represented by the siRNA-induced drug sensibilizationindex (DS) which corresponds to the cell population (%) that survived toa simple chemotherapy treatment but died in response to the sametreatment with a siRNA transfection. It was calculated as follows: (CIsiRNA GFP−CI siRNAx)/CI SiRNA GFP×100. Significant differences betweenseries were tested by ANOVA with Statview (SAS institute). Differenceswere considered significant when p<0.05 (*) and very significant whenp<0.01 (**).

d) Q-RT PCR

RNAs were extracted on silica column (Nucleospin< >, MACHEREY-NAGEL).RNAs were reverse transcribed using the M-MLV reverse transcriptaseRNase H minus enzyme (Promega) and oligodT as a primer, at 40 deg. C.during 1.5 hours. After reverse transcription, cDNAs were purified (MiniElute, PCR Purification Kit< >, QIAGEN) and assayed by quantitative PCRusing the HotStar Reaction mix (ROCHE), using a pair of primers specificfor the candidate gene and the reference gene ERCC1 (Table III).Measurements were carried out on a Light Cycler thermo-cycler (ROCHE).

TABLE III qRT-PCR primers sequences Annealing product SEQ temperaturesize Gene Primer Sequence (5′-3′) ID NO: (deg. C.) (bp) Cyclophyline AForward ttcatctgcactgccaagac 1 61.8 158 Reverse tcgagttgtcacagtcagc 2ERCC1 Forward ggcgacgtaattcccgacta 3 61.8 121 Reverseagttcttccccaggctctgc 4 ERCC2 Forward cggaactatgggaacctcct 5 64.0 200Reverse tacttctccagggcgacact 6 MUTYH Forward gtcctgacgtggaggagtgt 7 64.0200 Reverse cctctgcaccagcagaattt 8 PNKP Forward tcgagagatgacggactcct 964.0 206 Reverse tttattgtggaggggagctg 10

Measures were performed twice from each cell sample, and results wereexpressed as the mean of at least three independent samples (originatingfrom independent in vitro experiments).

2) Results a) Identification of DNA Repair Genes Potentially Involved inChemoresistance of Astrocytoma Cells

Genes potentially responsible for chemoresistance of astrocytoma, werescreened from the chromosomic area commonly considered as correlatedwith oligodendroglioma chemosensitivity (1p36-1p32 and 19q13.2-19q13.4;Smith et al., Oncogene, 1999; 18, 4144-4152). Gene sequences wereretrieved from data bases on the web(http://www.ncbi.nlm.nih.gov/PubMed/; http://www.ensembl.org;http://www.cgal.icnet.uk/DNA Repair Genes.html; Supplement to the paperby Wood et al., Mutat. Res., 2005, 577, 275-283) and published data(Harris, A. L., Int J Radiat Biol Relat Stud Phys Chem Med, 1985, 48,675-90; Christmann et al., Toxicology, 2003, 193, 3-34; Wood et al.,Mutat. Res., 2005, 577, 275-283). Eight genes belonging to differentrepair systems were selected (Table IV).

TABLE IV List of DNA repair-associated genes located 1p/19q LOH regionsDNA repair genes DNA repair system Accession number Localization LIG1NER NM_000234 19q13.2-3 ERCC2 (XPD) NER NM 000400 19q 13.3 POLD1 NER andMMR** NM_002691 19q13.3 RUVBL2 Homologous NM_006666 19q13.3Recombination PNKP BER*** NM_007254 19q13.3-4 RAD 54L HomologousNM_003579 1p32 Recombination MUTYH BER NM_012222 1p34.3 MAD2L2 DNApolymerase NM 006341 1p36 (REV7) *NER: Nucleotide Excision Repair **NMR:Nucleotide Mismatch repair ***BER: Base excision repair

b) In Vitro Chemosensitivity Assay

An in vitro assay for the siRNAs high-throughput screening, wasdeveloped to find out which genes (Table IV) are implicated in gliomachemoresistance mechanisms. The cell number after chemotherapy and siRNAtransfection was measured to have a global chemosensitizationevaluation, defined as a significant increased cell death compared withthe effect of the drug alone. In a first series of experiments, thefollowing conditions of the assay were established: (i) drug and siRNAtreatment protocols, (ii) specific temporal sequence of cell seeding anddrug treatment, (iii) siRNA transfection, (iii) cell viabilitymeasurement. CDDP was chosen because it is an alkylating agent harboringa very reproducible activity in vitro. Most, if not all, cell damagesoccurred during the first hour of drug treatment since one and 24hour(s) incubations were equally efficient. Moreover, it was establishedthat the mRNA amount was the lowest 24 and 48 hours after siRNAtransfection. Altogether, these data suggested that siRNAs had to beadded prior to the drug.

c) Screening of DNA-Repair Genes Involved in Chemoresistance ofAstrocytoma Cells

The screening was performed in three steps. First siRNAs targetingcandidate genes were screened on one cell line and results wereconfirmed at statistic level in further experiments. The study was thenextended to six astrocytoma-derived cell lines and the siRNAs werevalidated at molecular level. Finally, the study was extended to asecond chemotherapy agent.

The eight DNA-repair genes located in the 1p/19q (Table IV) werescreened with five different siRNAs by gene, on the GHD cell line. ThesiRNAs sequences are presented in Table V. One siRNA specific for GFPand one siRNA specific for ERCC1 were used as controls.

TABLE V SiRNAs sequences Gene siRNA sequence*(1) (2) SEQ ID NO: ERCC25′-ggacgucgaugggaaaugc-3′ 11 5′-agacggugcucaggaucaa-3′ 125′-ucaucaucgagcccuuuga-3′ 13 5′-ggaacaagcugcucuuuau-3′ 145′-ugacuuucuuaccuucgau-3′ 15 LIG1 5′-agacgcucagcagcuucuu-3′ 165′-gaagauagacaucaucaaa-3′ 17 5′-agacagcagagcccagaaa-3′ 185′-gcagacguucugcgagguu-3′ 19 5′-gcagauccagccauuccaa-3′ 20 MAD2L25′-gaagaaugauguggagaaa-3′ 21 5′-gacucgcuguugucucaug-3′ 225′-cucgcaacaugcagaagau-3′ 23 5′-gaagauccaggucaucaag-3′ 245′-ugagcaggauguccacaug-3′ 25 MUTYH 5′-gaagcaugcuaagaacaac-3′ 265′-ugggaugauugcugagugu-3′ 27 5′-gcacccuuguuucccagca-3′ 285′-gguuguccacaccuucucu-3′ 29 5′-gcugacauaucaaguauau-3′ 30--> PNKP5′-cacacuguauuuggucaau-3′ 31 5′-agagacccgcacaccagaa-3′ 325′-gaaucuuguacccagagau-3′ 33 5′-aguccaccuuucucaagaa-3′ 345′-caaccgguuucgagagaug-3′ 35 POLD1 5′-ggagauggaggcagaacac-3′ 365′-guuggagauugaccauuau-3 37 5′-ucaccgguuacaacaucca-3′ 385′-cuuagacuccaccagcugc-3′ 39 5′-auucagaugggauaccucc-3′ 40 RAD54L5′-ccagcauugugaauagaug-3′ 41 5′-ucaccucgcuaaagaagcu-3′ 425′-ggagcuguuuauccuggau-3′ 43 5′-ugaucugcuugaguauuuc-3′ 445′-gcagugagacccagaucca-3′ 45 RUVBL2 5′-aucuucucccuggagauga-3′ 465′-acugacccucaagaccaca-3′ 47 5′-acgcaaggguacagaagug-3′ 48 GFP5′-gacguaaacggccacaaguuc-3′ 49 ERCCI 5′-ggagcuggcuaagaugugu-3′ 50 (1)siRNA sequences are defined by the sequence of the sense strand. Theantisense strand is perfectly complementary to the sense strand. SiRNAsresponsible for a significant sensitization effect are in bold. (2) Saidsequences comprise a 3′-terminal tt overhang.

Cell viability was measured in absence and in presence of CDDP. SiRNAswere selected on the basis of two criteria: the absence of basaltoxicity and their efficiency to improve chemotherapy treatment. ToxicsiRNAs inducing more than 35% cell death after transfection (withoutCDDP) were discarded. In contrast, those increasing cell death wereretained. An siRNA increased chemosensitivity when its chemoresistanceindex (CI) was lower than GFP siRNA CI, ie lower than 0.5. In FIG. 1,the white square corresponds to siRNA with low toxicity and withchemo-sensitization properties. Eight siRNAs out of 38 matched withthese criteria, corresponding to 5 genes: ERCC2, MUTYH, PNKP, POLD1, andRUVBL2 (FIG. 1). Only one sequence of MUTYH, PNKP, RUVBL2 and POLD1 (SEQID NO: 30, 31, 38 and 46) siRNAs increased chemosensitivity while 4 (SEQID NO: 11, 13, 14 and 15) out of 5 sequences targeting ERCC2 fulfilledthese conditions.

The most efficient siRNA for each of these genes was transfected in 6different glioma derived cell lines treated with CDDP or temozolomide(TMZ). CIs for each siRNA were compared with control CI (siRNA GFP). Theresults confirmed that three siRNAs targeting ERCC2, MUTYH and PNKP,respectively, had a significant chemo-sensitization effect onastrocytoma cells (FIG. 2). The three siRNAs were equally efficient (upto 17%) on 3 (ERCC2) or 2 (MUTYH and PNKP) cell lines (FIG. 2). Whencombined by 2 or 3, siRNAs did not show any greater efficiency. Bycomparison, inhibition of ERCC1 was more potent to sensitize cells toCDDP (up to 24.9%) and its effect was the most widespread since 4 out ofthe 6 cell lines were sensitized.

The previously observed effects were validated at molecular level; asignificant mRNA content decrease was confirmed by qRT-PCR for allsiRNAs (Table VI).

TABLE VI Validation of SiRNA- Cell line mRNA inhibition % PANOVA GHDERCC2 61.6 0.011 MUTYH 71.0 0.004 PNKP 83.8 0.005 ERCC1 58.6 0.019 U373ERCC2 95.7 0.003 MUTYH 72.9 0.007 PNKP 44.2 0.013 ERCC1 77.4 0.018

When a particular siRNA was transfected, the quantities in mRNAcorresponding to all the other non targetted genes were unchanged,confirming the target specificity of the selected siRNA. There was noobvious link between the efficiency of siRNA to decrease mRNA amounts(nor with final mRNA content) and functional impact on cell viability(FIG. 2). This is reinforced by the observation that ERCC1 mRNA amountactually decreased (by 65%) in U87 cells which were yet notchemosensitized.

d) Study for a Link Between DNA-Repair Genes Expression andsiRNA-Induced Chemotherapy

Expression of the 4 DNA-repair genes was studied by qRT-PCR in the 6cell lines, with (induced level) or without cisplatin (basal level) toanalyse the hypothesis that differences in expression could account fordifferences in siRNA responses. For each individual mRNA, the relativegene expression levels were roughly similar in all cell lines in absenceof drug (Table VII).

TABLE VII DNA repair gene expression Cell Basal Induced line Qty* semQty sem %** ERCC1 GHD 33 13 35 15 104 LN229 33 13 78 30 233 U138 40 1797 36 242 U373 11 5 12 6 110 U87 25 10 25 10 99 CCF 8 4 12 6 148 ERCC2GHD 10 3 17 5 163 LN229 4 1 11 1 268 U138 21 4 66 15 312 U373 14 1 17 4122 U87 5 1 5 1 96 CCF 2 1 3 1 196 MUTYH GHD 108 19 114 14 105 LN229 13752 439 44 320 U138 185 43 469 157 254 U373 49 33 135 41 278 U87 91 28101 37 110 CCF 45 20 117 40 257 PNKP GHD 94 28 83 23 88 LN229 137 35 28370 206 U138 550 116 1198 171 218 U373 115 31 149 4 130 U87 224 50 196 6388 CCF 76 20 130 29 171 *quantity in zeptomol (10<-21> mol) in a 25 ngtotal RNA extract **percentage of basal level

MUTYH and PNKP mRNAs were the most abundant and ERCC2 was generally theless one. Expression of the four genes was reinforced up to three timesin response to chemotherapy, in at least two cell lines (Table VII). Nogene was up-regulated in all cell lines, the four genes wereup-regulated in two cell lines (LN229 and U138) and none in one other(U87). Thus, there was no common induction profile conserved among allcell lines. Finally, no correlation was found between chemosensitizationby a siRNA (DS) and basal or CDDP-induced expression of correspondinggene or even global expression of the 4 genes.

e) Transposition of Chemo-Sensitization by siRNAs from CDDP toTemozolomide.

Since temozolomide is becoming the chemotherapy gold standard forgliomas, the study was extended to this drug. ERCC1 siRNAs were moreefficient for sensitizing astrocytoma-derived cell lines to this seconddrug. However, down-regulating MUTYH, PNKP or ERCC2 improved verysignificantly drug effects in U373 cells (FIG. 2).

EXAMPLE 2 siRNA Directed to 1p/19q Genes Involved in Drug Metabolism areAble to Sensitize Cancer Cells to Chemotherapy 1) Material and Methods

The experimental procedures are described in example 1.

2) Results a) Identification of Genes Potentially Involved inChemoresistance of Astrocytoma Cells

Genes potentially responsible for chemoresistance of astrocytoma, werescreened from the chromosomic area considered as commonly correlatedwith oligodendroglioma chemosensitivity, as described in example 1. Ninegenes involved in drug metabolism (detoxification, cellular efflux,apoptosis) were selected (Table VIII).

TABLE VIII List of drug metabolism-associated genes located on 1p/19qLOH regions Accession Genes Function number Localization CYP2B6 Drug andlipid metabolism: Cytochrome P450 NM 000767 19q13.2 CYP2F1 proteins(monooxygenases) catalyse reactions NM 000774 19q13.2 CYP2A6 {closeoversize brace} involved in drug metabolism and synthesis NM_00076219q13.2 CYP4B1 of cholesterol, steroids and other lipids NM_0007791p34-p12 FRAP1 Signal transduction: The corresponding protein NM 0049581p36.2 belongs to the family of phosphatidylinositol kinase-relatedkinases. These kinases mediate cellular responses to stresses such asDNA damage and nutrient deprivation MGC13170 Multidrugresistance-related protein: Putative NM 199249 19q13.33 MDR-likefunction MLP Signal transduction: The myristoylated, alanine- NM_0230091p35.1 rich protein MARCKS is a widely expressed, prominent substratefor protein kinase C, involved in brain development MSH4 DNA metabolism:DNA mismatch repair and NM 002440 1p31 meiotic recombination RPS8Translation: Protein participating to translation NM 001012 1p34.1-p32complex

b) Screening of DNA-Repair Genes Involved in Chemoresistance ofAstrocytoma Cells

The eight genes located in the 1p/19q (Table VIII) were screened withfive different siRNAs by gene, on two different cell-lines, U373 andGHD. The siRNAs sequences are presented in Table IX. A siRNA specificfor GFP was used as control.

TABLE IX SiRNA sequences* po- SEQ Gene siRNA sequence sition ID NO:CYP2B6 ccaccauccuccagaacuu 1359 51 ggaaaucaaugcuuacauu 724 52acaggugauuggcccacau 994 53 ugacccacacuacuuugaa 1198 54acacgcucucgcucuucuu 879 55 ccagggagaggaguuuagu 326 91gaagcauugaggagcgaau 475 92 CYP2F1 ccacacauaaccugcucuu 928 56gcauaagcacagccaucuu 64 57 ucaaugacaacuuccaaau 661 58 acacggaguucuacuugaa857 59 ccaccgucaugcagaacuu 1370 60 CYP2A6 ugaccacguugaaccucuu 887 61ccaaguuucgggauuucuu 1142 62 gcaccagcaucguuguaga 1020 63ggacaaagaguuccuguca 591 64 gcaagccugucaccuuugu 1175 65 CYP4B1ugagccugacuaugccaaa 297 66 ugaugugcugaagcccuau 453 67ggagucuacucgcuucuau 253 68 gcacgaucauucuucucau 3228 69cuaccuugcagucagcgau 645 70 FRAP1 agaacucgcugauccaaau 1230 71ccagcagcauaagcaggaa 5287 72 caagcgacaucccaugaaa 7670 73gcaggcugcucuccauggu 969 74 ggcucaugcugggacccaa 1010 75--> MGC13170ggagcuguccauacgccac 1159 76 ggagaagguggauaagugg 1068 77ccaggcucaugcugggacc 1007 78 gugcagccucagaagaaga 674 103gaagaagaaauucucuuuc 453 104 MLP cgagggcacugcucaggaa 597 79gaagaaauucucuuucaag 456 80 aaagcaauggagacuuauc 293 81cgacuucguucuaauauau 1089 82 ucaacuuccuucagaauuu 1694 83 MSH4guagacgacuucguucuaa 1084 84 agagcuuacuaugguuccu 1380 85agaagguauuggcauuugu 2345 86 gcugacuccugaggaagaa 407 87ccaagacccuggugaagaa 301 105 RPS8 ccacaagaagcggaaguau 86 88agaguuggaguucuaucuu 602 89 agagaaagcccuaccacaa 73 90 *siRNA sequencesare defined by the sequence of the sense strand. The antisense strand isperfectly complementary to the sense strand. SiRNAs responsible for asignificant sensitization effect are highlighted in bold.

To select siRNAs specifically including a chemosensitive effect, thoseinducing a basal toxic effect were eliminated (more than 35% cell deathafter transfection). A positive chemosensitive effect was considered forsiRNAs inducing a cytotoxic effect compared to siRNA GFP/CDDP condition.2 siRNAs out of 45, matched with these criteria, corresponding to 2genes: CYP2A6 and CYP4B1 (Table IX).

The most efficient siRNA for each of these genes was transfected in 6different glioma derived cell lines treated with CDDP or temozolomide(TMZ). CIs for each siRNA were compared with control CI (siRNA GFP). Theresults confirmed that two siRNAs (SEQ ID NO: 64 and 70) targetingCYP2A6 and CYP4B1, respectively, had a significant chemosensitizationeffect on astrocytoma cells (FIG. 3).

1. A double-stranded short interfering nucleic acid molecule comprisinga sense region and an antisense region, wherein the sense regioncomprises a nucleotide sequence that is selected from the groupconsisting of: SEQ ID NO: 64, SEQ ID NO: 70, and sequences having atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity with SEQ ID NO: 64 or SEQ ID NO: 70, and theantisense region comprises a nucleotide sequence that is complementaryto the sense region.
 2. The double-stranded short interfering nucleicacid molecule according to claim 1, wherein each strand of the moleculecomprises 15 to about 30 nucleotides, and each strand of the moleculecomprises at least 15 to about 30 nucleotides that are complementary tothe nucleotides of the other strand.
 3. The double-stranded shortinterfering nucleic acid molecule according to claim 2, wherein themolecule comprises a 19 to 21-nucleotide duplex.
 4. The double-strandedshort interfering nucleic acid molecule according to claim 1, whereinthe sense region comprises the nucleotide sequence of SEQ ID NO: 64 orSEQ ID NO: 70, and the antisense region comprises the nucleotidesequence of SEQ ID NO: 101 or SEQ ID NO: 102, respectively.
 5. Thedouble-stranded short interfering nucleic acid molecule according toclaim 1, comprising 1 to about 3 overhanging nucleotides at the 3′ endof each strand.
 6. The double-stranded short interfering nucleic acidmolecule according to claim 1, comprising blunt end(s).
 7. Thedouble-stranded short interfering nucleic acid molecule according toclaim 1, wherein the molecule is assembled from two separateoligonucleotide fragments, and one fragment comprises the sense regionand a second fragment comprises the antisense region of the shortinterfering nucleic acid molecule.
 8. The double-stranded shortinterfering nucleic acid molecule according to claim 1, wherein thesense region is connected to the antisense region via a linker molecule.9. The double-stranded short interfering nucleic acid molecule accordingto claim 1, comprising ribonucleotides.
 10. The double-stranded shortinterfering nucleic acid molecule according to claim 1, comprising oneor more modified pyrimidine and/or modified purine nucleotides.
 11. Thedouble-stranded short interfering nucleic acid molecule according toclaim 1, wherein a strand comprising the sense region includes aterminal cap moiety at the 5′ and/or 3′-end(s).
 12. The double-strandedshort interfering nucleic acid molecule according to claim 1, wherein astrand comprising the antisense region includes a phosphate group at the5′-end.
 13. The double-stranded short interfering nucleic acid moleculeaccording to claim 1, comprising at least one modified inter-nucleotidelinkage.
 14. A transcription unit comprising: a transcription initiationregion; a transcription termination region; and a nucleic acid sequenceencoding at least one short interfering nucleic acid molecule accordingto claim 1, wherein said nucleic acid sequence is operably linked tosaid transcription initiation region in a manner that allows expressionand/or delivery of the short interfering nucleic acid molecule.
 15. Anexpression vector comprising a transcription unit comprising: atranscription initiation region; a transcription termination region; anda nucleic acid sequence encoding at least one short interfering nucleicacid molecule according to claim 1, wherein said nucleic acid sequenceis operably linked to said transcription initiation region in a mannerthat allows expression and/or delivery of the short interfering nucleicacid molecule.
 16. A cell modified by an expression vector comprising atranscription unit comprising: a transcription initiation region; atranscription termination region; and a nucleic acid sequence encodingat least one short interfering nucleic acid molecule according to claim1, wherein said nucleic acid sequence is operably linked to saidtranscription initiation region in a manner that allows expressionand/or delivery of the short interfering nucleic acid molecule.
 17. Apharmaceutical composition comprising at least: a short interferingnucleic acid molecule according to claim 1, or an expression vectorcomprising a transcription unit, said transcription unit comprising atranscription initiation region, a transcription termination region, anda nucleic acid sequence encoding at least one short interfering nucleicacid molecule according to claim 1, wherein said nucleic acid sequenceis operably linked to said transcription initiation region in a mannerthat allows expression and/or delivery of the short interfering nucleicacid molecule, in an acceptable carrier.
 18. A pharmaceuticalcomposition comprising at least: a short interfering nucleic acidmolecule according to claim 1, or an expression vector comprising atranscription unit, said transcription unit comprising a transcriptioninitiation region, a transcription termination region, and a nucleicacid sequence encoding at least one short interfering nucleic acidmolecule according to claim 1, wherein said nucleic acid sequence isoperably linked to said transcription initiation region in a manner thatallows expression and/or delivery of the short interfering nucleic acidmolecule, in an acceptable carrier, the composition comprising acombination of at least two different short interfering nucleic acidmolecules.
 19. A pharmaceutical composition comprising at least a shortinterfering nucleic acid molecule according to claim 1, or an expressionvector comprising a transcription unit, said transcription unitcomprising a transcription initiation region, a transcriptiontermination region, and a nucleic acid sequence encoding at least oneshort interfering nucleic acid molecule according to claim 1, whereinsaid nucleic acid sequence is operably linked to said transcriptioninitiation region in a manner that allows expression and/or delivery ofthe short interfering nucleic acid molecule, in an acceptable carrier,wherein the short interfering nucleic acid molecule or the expressionvector is associated with at least one anticancer drug.
 20. The shortinterfering nucleic acid molecule according to claim 1 as a medicament,in a form to be administered by a single route or by multiple routesselected from local, parenteral, oral, sub-lingual or inhalation. 21.The expression vector according to claim 15 as a medicament, in a formto be administered by a single route or by multiple routes selected fromlocal, parenteral, oral, sub-lingual or inhalation.
 22. A method fortreating cancer comprising administering to a patient in need thereof:the short interfering nucleic acid molecule according to claim 1, or anexpression vector comprising a transcription unit, said transcriptionunit comprising a transcription initiation region, a transcriptiontermination region, and a nucleic acid sequence encoding at least oneshort interfering nucleic acid molecule according to claim 1, whereinsaid nucleic acid sequence is operably linked to said transcriptioninitiation region in a manner that allows expression and/or delivery ofthe short interfering nucleic acid molecule.
 23. A product comprising atleast: a short interfering nucleic acid molecule according to claim 1,or an expression vector comprising a transcription unit, saidtranscription unit comprising a transcription initiation region, atranscription termination region, and a nucleic acid sequence encodingat least one short interfering nucleic acid molecule according to claim1, wherein said nucleic acid sequence is operably linked to saidtranscription initiation region in a manner that allows expressionand/or delivery of the short interfering nucleic acid molecule, and ananticancer drug, as a combined preparation for simultaneous, separate orsequential use in anticancer therapy.